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GC-MS analysis of ethyl acetate extract of

Sterptomyces species isolated from vermicast

Article in International Journal of Pharma and Bio Sciences · November 2016

DOI: 10.22376/ijpbs.2016.7.4.b416-419

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 Int J Pharm Bio Sci 2016 Oct ; 7(4): (B) 416 – 419

Original Research Article Microbiology

ISSN
International Journal of Pharma and Bio Sciences 0975-6299

GC- MS ANALYSIS OF ETHYL ACETATE EXTRACT

OF STERPTOMYCES SPECIES ISOLATED
FROM VERMICAST
R. BALACHANDAR1*, K. ASHOK KUMAR 2 AND N. KARMEGAM 3

*1Research Scholar, Faculty of Bio-Engineering, Sathyabama University, Chennai-600 119, Tamil Nadu, India
2
Arulmigu Meenakshi Amman College of Engineering, Vadamavandal, Namandi-604 410, Cheyyar Taluk, Near
Kanchipuram, Tamil Nadu, India
3
Department of Botany, Government Arts College (Autonomous), Salem-636 007, Tamil Nadu, India

ABSTRACT

In this study, the bioactive compounds present in the ethyl acetate extracts of two different Streptomyces
strains isolated from vermicast (earthworm fecal pellets) were investigated using Gas Chromatography
and Mass Spectrometry (GC-MS). GC-MS analysis showed the presence of 12 and 9 bioactive
compounds in AS1 and AS3 respectively. GC-MS analysis of AS1extract revealed the presence of major
bioactive compounds such as n-hexadecanoic acid, 6-hexadecanoic acid, methyl ester, 9-octadeconic
acid, methyl ester, 2-4, cycloheadien-1-one, 3,5-bis (1,1-dimethyl ethyl) 4-hydroxy and AS3 extract
contains hexadecanoic acid, 9-octadecanic acid-E, oleic acid, 6-ocatadecanic acid, 2-4, cycloheadien-1-
one, 3,5-bis (1,1-dimethyl ethyl) 4-hydroxy, 1,2-Benzisothiazol-3- amine tbdms, hexamethyl
cyclotrisiloxane and 1,2-Bis (trimethylsilyl) benzene.

KEYWORDS: GC-MS analysis, bioactive compounds, Streptomyces species, ethyl acetate extract, vermicast.

R. BALACHANDAR
Research Scholar, Faculty of Bio-Engineering, Sathyabama University,
Chennai-600 119, Tamil Nadu, India

*Corresponding author

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 Int J Pharm Bio Sci 2016 Oct ; 7(4): (B) 416 – 419
INTRODUCTION
Actinomycetes produce many important bioactive
compounds that have high commercial value. Their
ability to produce variety of bioactive compounds has
been utilized in a comprehensive series of researches in
numerous industries. Actinomycetes are universal
occurrence in nature and are widely distributed in
1 washed with water and counter stain safranine was
natural and man-made environments. The rhizosphere
soil is an ecological niche in which develop microbial
communities. Hatzinger and Alexander have shown that
bacteria that attained the highest densities in
rhizosphere survived successfully compared with those
1
at the lowest densities. Streptomyces are largely
isolated from soil and marine sediments. Although soils
has been screened by the pharmaceutical industry from
about 50yrs only a miniscule fraction of the surface of
the globe has been sampled, and only a small fraction of
2
Streptomyces has been discovered. Isolation of
Streptomyces from soil source may be valuable for the
production of antibiotics. Streptomyces species
syntheses many different biologically active secondary
metabolites such as antibiotics, herbicides, pesticides,
anti parasitic and enzyme inhibitors, Of which antibiotics
are more important therapeutically. Approximately one
third of known antibiotics have been isolated from
3
streptomyces. Streptomyces produces of many
secondary metabolites, which have different biological
activities, such as antibacterial, antifungal, antiparasitic,
antitumor, anticancer and immunosuppressive actions.
4 40 C.
In particular the genus Streptomyces is an important
group of actinomycetes because of its ability to produce
many types of secondary metabolites. Streptomyces are
the group of Gram positive filamentous bacteria which
are ubiquitous various natural environments.
Streptomyces are the most economically valuable to
produce chemically diverse metabolites with wide range
5
of biological activity. The purpose of this study is to
analyze bioactive compounds present in the ethyl
acetate extract of Streptomyces species isolated from
vermicast.
MATERIALS AND METHODS
Six Vemicast samples were collected from the Shevaroy
hills, Eastern Ghats, Tamil Nadu and were transported
aseptically in sterile plastic containers to the laboratory.
The samples were air dried one week and kept at 45°C
for 1 hr to prevent bacterial and fungal contamination.
One gram of each vermicast sample was subjected to
serial dilution method and they were cultured in by
spread plate technique on starch casein agar and
incubated at 37°C for 2 weeks. Selected colonies were
subjected to starch casein nitrate broth for further
growth.
CULTURE CHARACTERISTICS
Isolated Streptomyces species were used to study the
morphology of spore bearing hyphae with the entire
spore chain. This is was done by using cover slip
method in which individual cultures were transferred to
the base of cover slips buried in starch casein agar
medium.
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GRAM STAINING
Smear of cultures were taken in clean glass slide and
heated gently over a flame. The smear was covered
with a thin film of crystal violet for 1min and washed
gently in slow running tap water. Gram’s iodine solution
was flooded over the smear for 1min and washed with
tap water. Alcohol was used to decolorize the smear
until the violet colour ceased to flow away. The side was
added over the smear for 2min, then the slide was
washed, drained, air dried, and viewed under
microscope. The culture retaining the violet colour
5
indicated that it was Gram positive organism.
EXTRACTION OF BIOACTIVE COMPOUNDS
Based on the preliminary screening, the most potent
isolates was grown in starch casein nitrate broth as a
production medium for the extraction of crude
compounds. The active strains were inoculated
individually in starch casein nitrate broth and incubated
for 5-7 days in shaker incubator at 28°C. Then it was
centrifuged for 20 mins at 10,000rpm and the
supernatant collected was mixed with an equal volume
of ethyl acetate. Ethyl acetate was added to the filtrate
in the ratio of 1:1(v/v) and incubated in shaker for 1 hr
for complete extraction. The ethyl acetate phase
contains bioactive compounds was separated from the
aqueous phase. The extracted crude compounds were
dried to get dry powder by using heating mantle at
o 6
GC-MS ANALYSIS
Ethyl acetate extract of Streptomyces species were
analyzed by GC-MS method. GC-MS technique was
performed by using GC Shimadzu QP2010 system and
gas chromatograph interfaced to a Mass Spectrometer
(GC-MS) equipped with Elite-1 fused silica capillary
column. Helium gas (99.99%) was used as the carrier
gas at a constant flow rate of 1.51ml/min and an
injection volume of 2µl was employed (split ratio: 20).
Injector temperature was 200°C; Ion-source temperature
200°C. The oven temperature was programmed from
70°C (Isothermal for 2 min.) with an increase of 300°C
for 10 min. Mass spectra were taken at 70eV; a scan
interval of 0.5 seconds with scan range of 40 -1000 m/z.
Total GC running time was 35 min.
7
IDENTIFICATION OF COMPOUNDS
Identification of components Interpretation of mass
spectra of GC-MS was done using the database of
National Institute of Standards and Technology (NIST)
having over 62,000 patterns. The mass spectrum of the
unknown component was compared with the spectrum
of the known components stored in the NIST 08 library8.
The name, mass and structure of the components of the
8
test materials were ascertained.
RESULTS AND DISCUSSION
Five isolates were collected based on their different
colony morphology and colour variations. Among these
two isolates were selected and purified by repeated
streak on starch casein nitrate agar medium sub-culture
plates for active strain. The selected species were
 Int J Pharm Bio Sci 2016 Oct ; 7(4): (B) 416 – 419

inoculated in starch casein nitrate broth and cultured 4,cyclohexadien 1-one, 1,2-bis (methyl ethyl) 4-
14
and were tested for GC-MS analysis to find out bioactive hydroxy. AS3 also contains major compounds n-
9 10 11
compounds. GC-MS chromatogram of the ethyl acetate hexadecanoic acid , hexadecanoic acid, methyl ester ,
15 16,17
extract of Streptomyces AS 1(Figure 1) showed 12 9-octadecanic acid-(E) , oleic acid , 6-ocatadecanic
18
bioactive compounds and AS3 (Figure 2) showed 9 acid , 2-4,cyclohexadien-1-one, 3,5-bis (1,1-dimethyl
14 19
bioactive compounds. Table 1 shows the presence of ethyl)4-hydroxy , 1,2-Benzisothiazol -3- amine tbdm ,
20
bioactive compounds in AS1 and the major compounds cyclotrisiloxane, hexamethyl. The earlier studies by
10
were n- hexadecanoic acid , hexadecanoic acid, methyl other researchers clearly shows that the above
11 12
ester , 1,2-Bis(trimethylsilyl)benzene , 9-octadeconic mentioned bioactive compounds possess antimicrobial
13 13
acid, methyl ester , 9-12 octadeconic acid (z,z) , 2- activity.

Figure 1
GC MS Chromatogram of AS1 Strain

Figure 2
GC MS Chromatogram of AS3 Strain

TABLE 1
Bioactive compounds identified in the Ethyl Acetate Extract of Streptomyces
isolates (AS1-AS3) by GC-MS.

S. No. Retention Time Compound Name Molecular Formula Mol. Weight

1 15.47 n- hexadecanoic acid C16H32O2 256
2 14.78 hexadecanoic acid, methyl ester C17H34O2 270
3 16.48 9-octadeconic acid, methyl ester (E) C19H36O2 296
4 16.85 9-12 octadeconic acid (z,z) C18H32O2 280
5 21.34 2-4,cyclohexadien 1-one, 3,5-bis (1,1-dimethyl ethyl) 4-hydroxy C14H22O2 222
6 16.85 oleic acid C18H34O2 282
7 16.85 6-ocatadecanic acid C18H34O2 282
8 19.66 1,2-Benzisothiazol -3- amine tbdms C12H22Si2 222
9 21.32 cyclotrisiloxane, hexamethyl C6H18O3Si3 222
10 21.34 1,2-Bis (trimethylsilyl) benzene C12H22Si2 222

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 Int J Pharm Bio Sci 2016 Oct ; 7(4): (B) 416 – 419
CONCLUSION
This study shows that AS1 and AS3 may possess
antimicrobial activity against various microorganisms
because of presence of potent antimicrobial
compounds. Thus the organisms studied can be used
as a potent tool for formulation of useful drugs. The
ethyl acetate extracts of that organisms shows the
REFERENCES
1. Duraipandiyan V, Sasi AH, Islam VI, Valanarasu
M, Ignacimuthu S. Antimicrobial properties of
actinomycetes from the soil of Himalaya. Journal
de Mycologie Médicale/Journal of Medical
Mycology. 2010 Mar 31;20(1):15-20.
2. Gesheva V. Rhizoshere microflora of some citrus
as a source of antagonistic actinomycetes.
European Journal of Soil Biology. 2002 Feb
28;38(1):85-8.
3. Usha Nandhini S and Masilamani M, Bioactive
Compounds Produced by Streptomyces Strain.
International J of Pharmacy and Pharamaceutical
Sciences.2013 Jan 5:176-178.
4. Bizuye A, Moges F, Andualem B. Isolation and
screening of antibiotic producing actinomycetes
from soils in Gondar town, North West Ethiopia.
Asian Pacific Journal of Tropical Disease. 2013
Oct 31;3(5):375-81.
5. Usha Nandhini S and Masilamani M, Bioactive
Compounds Produced by Streptomyces Strain.
International J of Pharmacy and Pharamaceutical
Sciences.2013 Jan 5:176-178.
6. Lalitha P,Karikalan K. and
Thirugnannsambantham K. Antimicrobial activity
of crude ethyl acetate extract of marine
Actinomycetes isolated form marine sediments.
International J of Current Research. 2013
Dec.5(12) :4053-4056.
7. Rana S, Salam MD. Antimicrobial potential of
actinomycetes isolated from soil samples of
Punjab. India. J Microbiol Exp. 2014;1(2):00010.
8. Sudar S, Justin Koil Pillai, GC-MS Analysis Of
Petroleum Ether And Methanol Extracts Of
Solanum Virginianum L Leaves. Int J Pharm Bio
Sci. 2014 Oct; 5(4): (P) 561 - 568.
9. Aruna S, Vijayalakshmi K, Shashikanth M,
Surekha R, Jyothi K. First report of antimicrobial
spectra of novel strain of Streptomyces tritolerans
(Strain AS1) isolated from earthworm gut (Eisenia
foetida) against plant pathogenic bacteria and
fungi. Curr Res Bacteriol. 2008;1:46-55..
10. Kalaivani CS, Sathish SS, Janakiraman N,
Johnson M. GC-MS studies on Andrographis
paniculata (Burm. f.) Wall. ex Nees–A medicinally
important plant. Int J Med Arom Plants. 2012
Feb;2(1):69-74.
11. Chandrasekaran M, Senthilkumar A, Venkatesalu
V. Antibacterial and antifungal efficacy of fatty
acid methyl esters from the leaves of Sesuvium
portulacastrum L. European review for medical
This article can be downloaded from www.ijpbs.net
B - 419
View publication stats
presence of bioactive compounds that possess
antimicrobial activity which can be eluted and purified
and can be used as a drug that can be assessed in
future studies.
CONFLICT OF INTEREST
Conflict of interest declared none.
and pharmacological sciences. 2011
Jul;15(7):775-80.
12. Tyagi R and Sharma V.GC-MS LB. A comparison
of volatile compounds in different genotypes of
sesamum indicum L. BY GC-MS. International J
of Pharma Sciences and Research.2014: 5(1):
249-258.
13. Wei LS, Wee W, Siong JY, Syamsumir DF.
Characterization of anticancer, antimicrobial,
antioxidant properties and chemical compositions
of Peperomia pellucida leaf extract. Acta Medica
Iranica. 2011 Oct 1;49(10):670
14. Wafaa H. B. Hassan, Ali A. El Gamal, Ebtesam
El-Sheddy, Mai Al-Oquil and Nida Nayyar
Farshori, The chemical composition and
antimicrobial activity of the essential oil of
Lavandula coronopifolia growing in Saudi Arabia.
J of Chem. Pharma. Res.2014: 6(2): 604-615.
15. Seanego CT, Ndip RN. Identification and
antibacterial evaluation of bioactive compounds
from Garcinia kola (Heckel) Seeds. Molecules.
2012 May 31;17(6):6569-84.
16. Choi JS, Park NH, Hwang SY, Sohn JH, Kwak I,
Cho KK, Choi IS. The antibacterial activity of
various saturated and unsaturated fatty acids
against several oral pathogens. Journal of
Environmental Biology. 2013 Jul 1;34(4):673.
17. Awa EP, Ibrahim S, Ameh DA. GC/MS analysis
and antimicrobial activity of diethyl ether fraction
of methanolic extract from the stem bark of
Annona senegalensis pers. International Journal
of Pharmaceutical Sciences and Research. 2012
Nov 1;3(11):4213.
18. Nahar N, Rahman MS, Rahman SM,
Moniruzzaman M. GC-MS analysis and
antibacterial activity of Trigonella foenum-
graecum against bacterial pathogens. Free
Radicals Antioxid. 2016;6:109-4.
19. Priyanka C, Kumar P, Bankar SP, Karthik L. In
vitro antibacterial activity and gas
chromatography–mass spectroscopy analysis of
Acacia karoo and Ziziphus mauritiana extracts.
Journal of Taibah University for Science. 2015
Jan 31;9(1):13-9.
20. Keskın D, Ceyhan N, Uğur A, Dbeys AD.
Antimicrobial activity and chemical constitutions of
West Anatolian olive (Olea europaea L.) leaves.
Journal of Food, Agriculture & Environment. 2012
Apr;10(2):99-102.